PROJECT SUMMARY The objectives of this project are 1) to develop cell-specific drug delivery systems; 2) to construct multifunctional drug delivery systems; and 3) to demonstrate therapeutic efficacy of these systems in animal models for treating genetic disorders, infectious diseases, as well as cancers. Cell-specific delivery in vivo is one of the most challenging issues in the field of drug delivery. To address this issue, we propose two strategies: (1) to mimic the receptor-mediated virus entry. The simple structure and composition of viruses make them capable of specifically infecting many cell types in humans. Our research program will apply knowledge of viral infections in order to guide the design of cell-specific delivery systems. For example, nucleolin has been identified as a cellular receptor on epithelial cells for human respiratory syncytial virus (RSV) in the lungs. We will utilize anti-nucleolin antibody or nucleolin ligands in order to target lung epithelial cells. (2) to target tumor over-expressed receptors. Previous studies, however, have identified a number of tumor over-expressed receptors, of which many are relatively unexplored and our understanding of them is very limited. For example, Chlorotoxin, a short peptide, is capable of crossing the blood brain barrier (BBB) and binding specifically to the matrix metalloproteinase-2 (MMP-2), which is over-expressed in brain tumor cells. We propose to utilize these ligands in order to specifically target tumor cells. We will evaluate their tumor specificity with imaging probes. In order to develop multifunctional drug delivery systems, we will integrate the ligands identified from the above studies with different delivery systems such as nanoparticles and conjugates. The payloads can be mRNA, siRNA, and small-molecule drugs to yield multifunctional delivery systems. The delivery systems are capable of utilizing mRNA for expressing functional proteins, siRNA for gene silencing, and small-molecule drugs for cancer therapy. By delivering mRNA encoding Cas9 and specific guide RNA (sgRNA), we anticipate achieving gene engineering in vivo in order to find a cure for genetic disorders such as hemophilia and to deplete viral genome for treating viral infections such as hepatitis B. In addition, we have designed Chlorotoxin-Cyanine-Drug (CCD) as a multifunctional conjugate in order to specifically deliver near- infrared molecular probes and cytotoxic agents to brain metastatic breast cancer (BMBC) tumor cells. After administration, CCD conjugates will cross the BBB and bind with MMP-2 on BMBC cells, triggering the cellular uptake of the CCD. Cyanine probes will facilitate imaging-guided tumor resection. Meanwhile, cytotoxic agents will induce the apoptosis of tumor cells, which are below detection limits of imaging and for which surgical removal is not a possibility. Our research goal is to translate the innovation of this research strategy in order to develop better drug delivery tools to treat diverse diseases.